About 4 billion cases of diarrhea occur annually and cause 4% of all deaths worldwide. Cholera caused by Vibrio cholerae and watery diarrhea caused by enterotoxigenic Escherichia coli (ETEC) cause substantial morbidity and mortality worldwide, and ETEC accounts for up to 20% of diarrhea cases in developing countries. The obstacles to controlling cholera and ETEC diarrhea in developing countries by public health and personal sanitation measures alone are formidable. No vaccine against cholera or ETEC diarrhea is approved in the United States, and cholera vaccines used elsewhere have significant limitations. Cholera toxin (CT) and the closely related type I heat-labile enterotoxin (LTI) cause watery diarrhea and are major virulence factors of V. cholerae and ETEC, respectively. V. cholerae and ETEC produce unrelated pili that have essential roles in their colonization of the small intestine, and antibodies against specific pilus components can block colonization and prevent diarrhea. Anti-CT or anti-LTI antibodies alone do not prevent diarrhea, but they are believed to act synergistically with antibodies that block colonization to provide enhanced protection against cholera and ETEC diarrhea, respectively. Our long-term goals are to: 1) understand at the molecular level the functions of CT and LTI, and 2) to develop safe and effective vaccines against V. cholerae and ETEC infections of humans. During the next project period, we will continue to study fundamental aspects of cholera toxin structure and function. We will investigate how the catalytically active CTA1 fragment recognizes its physiological substrate Gsa, what factors determine the functional consequences of interactions between CTA1 and its allosteric activator ADP ribosylation factor 6 (ARF6), and how monovalent vs. multivalent binding of ganglioside GM1 receptors by the pentameric B subunit affects the interactions of CT with target cells. We recently developed novel methods to make CT-based or LT-based enterotoxin-adhesin chimeras and demonstrated potent mucosal immunogenicity of such chimeras containing pilus tip adhesin proteins from ETEC. During the next project period, we will make and test a variety of enterotoxin-adhesin chimeras as candidate vaccines against cholera and ETEC diarrhea, and we will perform preliminary experiments to apply these technologies for the development of enterotoxin-adhesin chimeras as candidate vaccines against urinary tract infections caused by Escherichia coli and Proteus mirabilis. Our proposed studies on enterotoxin-adhesin chimeras as vaccine candidates represent applications of fundamental knowledge about enterotoxin structure and function that has been acquired over several decades for development of effective vaccines against bacterial infections of the intestinal tract and the urinary tract that are major world health problems.